Characterization of human aldehyde oxidase: substrate specificities, mode of inhibition and superoxide production

人醛氧化酶的表征：底物特异性、抑制模式和超氧化物产生

• 批准号: 224728554
• 负责人: Professorin Dr. Silke Leimkühler
• 金额: --
• 依托单位: Arbeitsgruppe für Molekulare Enzymologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/224728554?language=en
• 关键词: Characterization; human; aldehyde; oxidase; substrate

Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. So far, the physiological function of AOX for mammals is unknown. However, human AOX1 is of increasing pharmacological interest due to its recognized role in phase I drug metabolism. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. We established a codon-optimized heterologous expression system for human AOX1 in Escherichia coli. This expression system now enables us to obtain sufficient amounts of active protein for kinetic characterizations and sets the basis for site-directed mutagenesis and structure-function studies. To define the physiological function of human AOX1 the active site will be investigated in detail. We plan to characterize selected single polynucleotide polymorphisms (SNPs) identified around the substrate and inhibitor binding site in human individuals and analyze their effect on hAOX1 activity. We will further introduce amino acid exchanges around the FAD site and will analyze the effect on intramolecular electron transfer. Further, we will analyze the role of hAOX1 in the production of reactive oxygen species (ROS), since hAOX1 as a true oxidase was suggested to significantly contribute to the production of high amounts of endogenous O2-. The co-crystal structures of hAOX1 with substrates and inhibitors will be solved and will help to understand the mechanism of substrate and inhibitor binding for future drug developments. Our studies will be particularly important to define the physiopathological functions of AOX1 and will help to understand the drug metabolizing role of AOX1 for humans in general.

哺乳动物醛氧化酶（AOX）是一种存在于多种哺乳动物物种（包括人类和啮齿动物）的许多组织中的黄素化酶。不同物种含有不同数量的AOX同种型。到目前为止，AOX对哺乳动物的生理功能尚不清楚。然而，由于其在I相药物代谢中的公认作用，人AOX 1越来越受到药理学关注。特别是，哺乳动物而不是人类表达多种组织特异性AOX酶的原因是未知的。我们建立了一个密码子优化的人AOX 1在大肠杆菌中的异源表达系统。该表达系统现在使我们能够获得足够量的活性蛋白进行动力学表征，并为定点诱变和结构-功能研究奠定了基础。为了确定人AOX 1的生理功能，将详细研究活性位点。我们计划表征选定的单核苷酸多态性（SNP）周围的底物和抑制剂结合位点在人类个体中确定，并分析其对hAOX 1活性的影响。我们将进一步介绍FAD位点周围的氨基酸交换，并分析其对分子内电子转移的影响。此外，我们将分析hAOX 1在活性氧（ROS）产生中的作用，因为hAOX 1作为一种真正的氧化酶被认为是显着有助于产生大量的内源性O2-。hAOX 1与底物和抑制剂的共晶结构将得到解决，并将有助于了解底物和抑制剂结合的机制，为未来的药物开发提供帮助。我们的研究对于确定AOX 1的病理生理功能特别重要，并将有助于了解AOX 1对人类的药物代谢作用。